Magnesium promotes tea plant growth via enhanced glutamine synthetase-mediated nitrogen assimilation.

Acidic tea (Camellia sinensis) plantation soil usually suffers from magnesium (Mg) deficiency, and as such, application of fertilizer containing Mg can substantially increase tea quality by enhancing the accumulation of nitrogen (N)-containing chemicals such as amino acids in young tea shoots. However, the molecular mechanisms underlying the promoting effects of Mg on N assimilation in tea plants remain unclear. Here, both hydroponic and field experiments were conducted to analyze N, Mg, metabolite contents, and gene expression patterns in tea plants. We found that N and amino acids accumulated in tea plant roots under Mg deficiency, while metabolism of N was enhanced by Mg supplementation, especially under a low N fertilizer regime. 15N tracing experiments demonstrated that assimilation of N was induced in tea roots following Mg application. Furthermore, weighted gene correlation network analysis (WGCNA) analysis of RNA-seq data suggested that genes encoding glutamine synthetase isozymes (CsGSs), key enzymes regulating N assimilation, were markedly regulated by Mg treatment. Overexpression of CsGS1.1 in Arabidopsis (Arabidopsis thaliana) resulted in a more tolerant phenotype under Mg deficiency and increased N assimilation. These results validate our suggestion that Mg transcriptionally regulates CsGS1.1 during the enhanced assimilation of N in tea plant. Moreover, results of a field experiment demonstrated that high Mg and low N had positive effects on tea quality. This study deepens our understanding of the molecular mechanisms underlying the interactive effects of Mg and N in tea plants while also providing both genetic and agronomic tools for future improvement of tea production.

Aluminum Supplementation Mediates the Changes in Tea Plant Growth and Metabolism in Response to Calcium Stress.

Tea plants are more sensitive to variations in calcium concentration compared to other plants, whereas a moderate aluminum concentration facilitates the growth and development of tea plants. Aluminum and calcium show a competitive interaction with respect to the uptake of elements, consequently exerting physiological effects on plants. To further explore these interactions, in this study, we used the solution culture method to treat tea plants with two calcium concentrations (0.8 mM and 5.6 mM) and three aluminum concentrations (0 mM, 0.4 mM, and 1 mM). We then determined the influence of the combined treatments on root growth and quality compound accumulation in the tissues by a combination of phenotype, gene expression, and metabolite analyses. Moderate aluminum supplementation (0.4 mM) alleviated the inhibition of root growth caused by high calcium stress. High calcium stress significantly inhibited the accumulation of most amino acids (e.g., Glutamic acid, Citulline, and Arginine) and organic acids (e.g., a-ketoglutaric acid) in the roots, stems, and leaves, whereas aluminum deficiency significantly increased most amino acids in the roots and leaves (except Serine, Alanine, and Phenylalanine in the roots and Ser in the leaves), with a more than two-fold increase in Arg and Lysine. High calcium stress also induced the accumulation of secondary metabolites such as epigallocatechin gallate and procyanidin in the roots, whereas aluminum supplementation significantly reduced the contents of flavonol glycosides such as quercetin, rutin, myricitrin, and kaempferitrin, as well as caffeine, regardless of calcium concentration. Aluminum supplementation reversed some of the changes in the contents of leaf metabolites induced by calcium stress (e.g., 4-dihydroquercetin, apigenin C-pentoside, phenethylamine, and caffeine). Overall, calcium stress caused severe growth inhibition and metabolic disorders in tea plants, which could be reversed by aluminum supplementation, particularly in maintaining the root tips and the accumulation of secondary metabolites. These results provide a theoretical basis for improving calcium-aluminum nutrient management to promote tea plant growth and quality.

Effect of Interactions between Phosphorus and Light Intensity on Metabolite Compositions in Tea Cultivar Longjing43.

Light intensity influences energy production by increasing photosynthetic carbon, while phosphorus plays an important role in forming the complex nucleic acid structure for the regulation of protein synthesis. These two factors contribute to gene expression, metabolism, and plant growth regulation. In particular, shading is an effective agronomic practice and is widely used to improve the quality of green tea. Genotypic differences between tea cultivars have been observed as a metabolic response to phosphorus deficiency. However, little is known about how the phosphorus supply mediates the effect of shading on metabolites and how plant cultivar gene expression affects green tea quality. We elucidated the responses of the green tea cultivar Longjing43 under three light intensity levels and two levels of phosphorus supply based on a metabolomic analysis by GC×GC-TOF/MS (Two-dimensional Gas Chromatography coupled to Time-of-Flight Mass Spectrometry) and UPLC-Q-TOF/MS (Ultra-Performance Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry), a targeted analysis by HPLC (High Performance Liquid Chromatography), and a gene expression analysis by qRT-PCR. In young shoots, the phosphorus concentration increased in line with the phosphate supply, and elevated light intensities were positively correlated with catechins, especially with epigallocatechin of Longjing43. Moreover, when the phosphorus concentration was sufficient, total amino acids in young shoots were enhanced by moderate shading which did not occur under phosphorus deprivation. By metabolomic analysis, phenylalanine, tyrosine, and tryptophan biosynthesis (PTT) were enriched due to light and phosphorus effects. Under shaded conditions, SPX2 (Pi transport, stress, sensing, and signaling), SWEET3 (bidirectional sugar transporter), AAP (amino acid permeases), and GSTb (glutathione S-transferase b) shared the same analogous correlations with primary and secondary metabolite pathways. Taken together, phosphorus status is a crucial factor when shading is applied to increase green tea quality.

[Status of Heavy Metal in Organic Fertilizers in Main Tea Growing Regions of China].

To provide guidance for the safe use of organic fertilizers and improve soil quality and tea safety, it is necessary to conduct systematic analyses of the heavy metal content of organic fertilizers applied in the main tea producing areas of China. In this study, we analyzed the heavy metal contents in organic fertilizer samples collected from 2017 to 2019. The risks of collected organic fertilizers from different areas and sources were calculated. The results showed that the average concentrations of ω(As), ω(Hg), ω(Pb), ω(Cd), ω(Cr), ω(Cu), ω(Zn), and ω(Ni) in the collected organic fertilizers were 4.60, 0.22, 27.1, 0.78, 27.9, 58.3, 250.1, and 16.3 mg·kg-1, respectively. According to the assessment standard in NY/T 525- 2021, the over-limit rates of As, Hg, Pb, Cd, and Cr were 6.19%, 1.33%, 4.42%, 4.42%, and 1.33%, respectively. With respect to the area, the qualified rates were 100% in Shaanxi, Jiangsu, Anhui, Fujian, and Guangxi; 80%-90% in Shandong, Zhejiang, Hubei, Sichuan, Yunnan, and Guangdong; and only 54.5% in Jiangxi. The qualified rates of sources were 100% in rapeseed cake, soybean cake, and pig manure; 95.8% in sheep manure; 91.7% in cow manure; 90.7% in chicken manure; 87.2% in manure of other animals; 82.4% in the mixture of plant and animal sources; 65.2% in other plant sources; and 63.6% in other sources. According to the recommended application rate, the accumulation rate of heavy metals in soil with pig manure, cow manure, chicken manure, and sheep manure would be much higher than that with rapeseed cake and soybean cake. The average accumulation rate of organic fertilizer from animal sources was 7-30 times higher than that from plant sources. Therefore, it is recommended to use rapeseed cake or soybean cake fertilizer in tea plantation and to increase the supervision of heavy metal accumulation in soil and tea in those high-risk areas.

Integration of Metabolomics and Transcriptomics Reveal the Mechanism Underlying Accumulation of Flavonols in Albino Tea Leaves.

Albino tea plants (Camellia sinensis) have been reported to possess highly inhibited metabolism of flavonoids compared to regular green tea leaves, which improves the quality of the tea made from these leaves. However, the mechanisms underlying the metabolism of catechins and flavonols in albino tea leaves have not been well elucidated. In this study, we analyzed a time series of leaf samples in the greening process from albino to green in a thermosensitive leaf-color tea mutant using metabolomics and transcriptomics. The total content of polyphenols dramatically decreased, while flavonols (such as rutin) were highly accumulated in albino leaves compared to in green leaves. After treatment with increasing environment temperature, total polyphenols and catechins were increased in albino mutant tea leaves; however, flavonols (especially ortho-dihydroxylated B-rings such as rutin) were decreased. Meanwhile, weighted gene co-expression network analysis of RNA-seq data suggested that the accumulation of flavonols was highly correlated with genes related to reactive oxygen species scavenging. Histochemical localization further demonstrated that this specific accumulation of flavonols might be related to their biological functions in stress tolerance. These findings suggest that the temperature-stimulated accumulation of total polyphenols and catechins in albino mutant tea leaves was highly induced by enhanced photosynthesis and accumulation of its products, while the initial accumulation and temperature inhibition of flavonols in albino mutant tea leaves were associated with metabolism related to oxidative stress. In conclusion, our results indicate that the biosynthesis of flavonoids could be driven by many different factors, including antioxidation and carbon skeleton storage, under favorable and unfavorable circumstances, respectively. This work provides new insights into the drivers of flavonoid biosynthesis in albino tea leaves, which will further help to increase tea quality by improving cultivation measures.

Dynamic Changes in the Antioxidative Defense System in the Tea Plant Reveal the Photoprotection-Mediated Temporal Accumulation of Flavonoids under Full Sunlight Exposure.

To reveal the mechanisms underlying how light affects flavonoid metabolism and the potential role of flavonoids in protecting against photooxidative stress in tea leaves, tea plants adapted to low-light conditions were exposed to full sunlight over 48 h. There was an increase in the activities of catalase (CAT) and superoxide dismutase (SOD) as well as greater accumulation of reactive oxygen species, lutein, tocopherols, ascorbate and malondialdehyde, suggestive of a time-dependent response to photooxidative stress in tea leaves. Analysis of the time dependency of each element of the antioxidant system indicated that carotenoids and tocopherols exhibited the fastest response to light stress (within 3 h), followed by SOD, CAT and catechin, which peaked at 24 h. Meanwhile, flavonols, vitamin C and glutathione showed the slowest response. Subsequent identification of the main phytochemicals involved in protecting against oxidative stress using untargeted metabolomics revealed a fast and initial accumulation of nonesterified catechins that preceded the increase in flavonol glycosides and catechin esters. Gene expression analysis suggested that the light-induced accumulation of flavonoids was highly associated with the gene encoding flavonol synthase. Ultraviolet B (UV-B) irradiation further validated the time-dependent and collaborative effects of flavonoids in photoprotection in tea plants. Intriguingly, the dynamics of the metabolic response are highly distinct from those reported for Arabidopsis, suggesting that the response to light stress is not conserved across plants. This study additionally provides new insights into the functional role of flavonoids in preventing photooxidative stress and may contribute to further improving tea quality through the control of light intensity.

Glutamate dehydrogenase isogenes CsGDHs cooperate with glutamine synthetase isogenes CsGSs to assimilate ammonium in tea plant (Camellia sinensis L.).

Glutamate dehydrogenase (GDH) is a central enzyme in nitrogen metabolism, assimilating ammonia into glutamine or deaminating glutamate into α-oxoglutarate. Tea (Camellia sinensis L.) plants assimilate ammonium efficiently, but the role of CsGDH in ammonium assimilation remains unclear. We confirmed that tea has three GDH isogenes: CsGDH1-3. Bioinformatic analysis showed that CsGDH1 encodes the ß-GDH subunit, CsGDH2/3 encode the α-GDH subunit, and their proteins all feature an NADH-specific motif. CsGDH1 is mainly expressed in mature leaves and roots, CsGDH3 is mainly expressed in new shoots and roots, and CsGDH2 has the highest expression level in flowers compared to the other five tissues. Expression patterns of CsGDHs and glutamine synthetase isogenes (CsGSs) under different ammonium concentrations suggested that CsGDHs cooperate with CsGSs to assimilate ammonium, especially under high ammonium conditions. Inhibition of GS and its isogenes resulted in significant induction of CsGDH3 in roots and CsGDH2 in leaves, indicating their potential roles in ammonium assimilation. Moreover, CsGDHs transcripts were highly abundant in chlorotic tea leaves, in constrast to those of CsGSs, suggesting that CsGDHs play a vital role in ammonium assimilation in chlorotic tea mutant. Altogether, our circumstantial evidence that CsGDHs cooperate with CsGSs in ammonium assimilation provides a basis for unveiling their functions in tea plants.

Metabolomics analysis reveals the responses of tea plants to excessive calcium.

BACKGROUND: The proper growth and development of tea plants requires moderately acidic soils and relatively low calcium levels, and excessive calcium at high pH can damage tea plant roots. To reveal the effects of calcium on the responses of tea plant to three pH levels (3.5, 5.0 and 6.5), a repeated test of two factors was designed. RESULTS: Root growth and elemental analysis indicated that excessive calcium improved the growth of tea roots at low pH conditions, whereas it did not harm the growth of tea roots under normal and high pH conditions, especially at pH 6.5. Excessive calcium antagonized the absorption and utilization of magnesium by tea plants. Gas chromatography-mass spectrometry results showed that the addition of Ca2+ resulted in the primary metabolism in roots being more active at a low pH level. By contrast, it had obvious adverse effects on the accumulation of root metabolites with high calcium treatment at normal or high pH. Differential metabolites identified using ultra-performance liquid chromatography quadrupole time of flight mass spectrometry indicated that flavonoids demonstrated the largest number of changes, and their biosynthesis was partially enriched with excessive calcium at low and high pH conditions, whereas it was down-regulated under normal pH conditions. Kaempferol 3-(2'-rhamnosyl-6'-acetylgalactoside) 7-rhamnoside, quercetin 3-(6'-sinapoylsophorotrioside) and delphinidin 3-(3'-p-coumaroylglucoside) showed the greatest increase. The results of gene expression related to root growth and calcium regulation were consistent with root growth and root metabolism. CONCLUSION: The overall results demonstrated that high Ca concentrations further aggravate the detrimental effects of high pH to tea roots. However, it is interesting that excessive calcium reduced the harm of a low pH on tea root growth to some extent. © 2021 Society of Chemical Industry.

Metabolomics reveals the within-plant spatial effects of shading on tea plants.

It is well known that green tea made from fully developed leaves located at the base of young shoots is of lower quality than that made from the still developing leaves located on the top of the shoot. It has additionally been shown that plant shading can significantly improve green tea quality. Here, we aimed to get more insight into the effects of shading on the overall metabolome in different parts of the tea shoots. To do this, field-grown tea plants were shaded by coverage with either a straw layer or a black net, both blocking the daylight intensity for more than 90%. Both the first (i.e. still developing) leaf and the fourth (i.e. fully developed) leaf, as well as the stem of young shoots were harvested and subjected to complementary untargeted metabolomics approaches, using accurate mass LC-Orbitrap-Fourier transform mass spectrometry (FTMS) for profiling both semi-polar and lipid-soluble compounds and GC-TOF-MS for profiling polar compounds. In total, 1419 metabolites were detected. Shading resulted in a decreased ratio of polyphenols to amino acids (which improves the quality of green tea) and lower levels of galloylated catechins in the shoots. The positive effect of shading on the amino acid/catechin ratio was more pronounced in the fully developed (fourth) than in the developing (first) leaves. Furthermore, many metabolites, especially organic acids, carbohydrates and amino acids, showed differential or opposite responses to the shading treatments between the three shoot tissues investigated, suggesting a within-plant spatial regulation or transport/redistribution of carbon and nitrogen resources between the tissues of the growing young shoots. This work provides new insight into the spatial effects of shading on tea plants, which could further help to increase tea quality by improving cultivation measures for plant shading.

Isolation and characterization of chloroplastic glutamine synthetase gene (CsGS2) in tea plant Camellia sinensis.

Tea plant (Camellia sinensis) is an ammonium preferring plant species. However, little is known about the mechanism underlying this preference. Herein, a chloroplastic glutamine synthetase gene (CsGS2), which is vital for nitrogen assimilation in mesophyll tissue, was isolated from tea cultivar C. sinensis cv. 'Longjing43'. The full length cDNA of CsGS2 was 1622 bp, having a 1299 bp open reading frame encoding a 432-amino acid protein. Homology search and sequence analysis demonstrated that CsGS2 protein carried the basic characteristics of a canonical GS2 domain and shared high identity with GS2s from other plant species. Subcellular localization and immunolocalization of CsGS2 revealed that it is localized in chloroplast. qRT-PCR and Western blot analyses showed that CsGS2 was expressed in a leaf-specific pattern, such that both CsGS2 and its protein were most abundant in mature leaves. Temporal expression patterns of CsGS2 showed minor differences in response to ammonium and nitrate nutrition. The transcript level of CsGS2 was significantly induced in mature leaves during the development of new shoots, whereas darkness inhibited this induction significantly. These results suggested that CsGS2 does not play a role in the differential utilization mechanisms of differing nitrogen forms in tea, and imply a light dependent transcription regulation in mature leaves during the development of new shoots.

Preferential assimilation of NH4+ over NO3- in tea plant associated with genes involved in nitrogen transportation, utilization and catechins biosynthesis.

Physiological effects of ammonium (NH4+) and nitrate (NO3-) on tea have confirmed that tea plants prefer NH4+ as the dominant nitrogen (N) source. To investigate the possible explanations for this preference, studies of 15NH4+ and 15NO3- assimilation using hydroponically grown tea plants were conducted. During the time course of 15NH4+ and 15NO3- assimilation, the absorption of 15N from 15NH4+ was more rapid than that from 15NO3-, as there was a more efficient expression pattern of NH4+ transporters compared with that of NO3- transporters. 15NH4+-fed tea plants accumulated more 15N than 15NO3- fed plants, which was demonstrated by that genes related to primary N assimilation, like CsNR, CsNiR, CsGDH and CsGOGAT, were more affected by 15NH4+ than 15NO3-. Markedly higher NH4+ concentrations were observed in 15NH4+-fed tea roots in comparison with NO3- treatment, whereas tea plants maintained a balanced concentration of NH4+ in tea leaves under both these two N forms. This maintenance was achieved through the increased expression of genes involved in theanine biosynthesis and the inhibition of genes related to catechins derived from phenylpropanoid pathway. The current results suggest that efficient NH4+ transportation, assimilation, and reutilization enables tea plant as an ammonium preferring plant species.

Stimulated biosynthesis of delphinidin-related anthocyanins in tea shoots reducing the quality of green tea in summer.

BACKGROUND: Greater proportions of purple tea buds and leaves usually appear in the summer, which seriously affects the color and taste quality of green tea products, yet the metabolism of purple tea shoots in summer remains unclear. Here, the metabolomic profiles and gene expression of related flavonoid metabolic pathways in the purple and normal green shoots of 'Longjing 43', and the quality of green tea made with these two phenotypes, were analyzed and compared. RESULTS: Differential metabolites identified using high-performance liquid chromatography-Orbitrap/mass spectrometry indicated that anthocyanin biosynthesis in purple leaves was enriched, with higher levels of anthocyanidins (delphinidin-hexose-coumaroyl showed the greatest increase), proanthocyanidins (oligomers of catechins) and kaempferol glycoside. Expression patterns of the genes ANR, ANS, FLS, LAR, C4H, PAL, CHI, CHS and DFR revealed that the metabolism of anthocyanin is positively regulated by high temperature and/or light levels in summer. Gas chromatography-mass spectrometry results showed that, in purple tea shoots, the metabolism of carbohydrates was enriched whereas that of amino acids was diminished, while their mannose, fructose, d-galactose, sorbose and d-glucose contents were more than double those found in green leaves. A sensory evaluation confirmed that a greater quantity of purple shoots had a greater negative impact on green tea quality because of a bitter taste and dark color (leaves and infusions were tested). CONCLUSIONS: These results highlight the need for and possibility of improving commercial tea quality via cultivation that controls the temperature or light of tea gardens during the summer. © 2019 Society of Chemical Industry.

Short-term inhibition of glutamine synthetase leads to reprogramming of amino acid and lipid metabolism in roots and leaves of tea plant (Camellia sinensis L.).

BACKGROUND: Nitrogen (N) nutrition significantly affected metabolism and accumulation of quality-related compounds in tea plant (Camellia sinensis L.). Little is known about the physiological and molecular mechanisms underlying the effects of short-term repression of N metabolism on tea roots and leaves for a short time. RESULTS: In this study, we subjected tea plants to a specific inhibitor of glutamine synthetase (GS), methionine sulfoximine (MSX), for a short time (30 min) and investigated the effect of the inhibition of N metabolism on the transcriptome and metabolome of quality-related compounds. Our results showed that GS activities in tea roots and leaves were significantly inhibited upon MSX treatment, and both tissue types showed a sensitive metabolic response to GS inhibition. In tea leaves, the hydrolysis of theanine decreased with the increase in theanine and free ammonium content. The biosynthesis of all other amino acids was repressed, and the content of N-containing lipids declined, suggesting that short-term inhibition of GS reduces the level of N reutilization in tea leaves. Metabolites related to glycolysis and the tricarboxylic acid (TCA) cycle accumulated after GS repression, whereas the content of amino acids such as glycine, serine, isoleucine, threonine, leucine, and valine declined in the MXS treated group. We speculate that the biosynthesis of amino acids is affected by glycolysis and the TCA cycle in a feedback loop. CONCLUSIONS: Overall, our data suggest that GS repression in tea plant leads to the reprogramming of amino acid and lipid metabolic pathways.

Characterization of three different classes of non-fermented teas using untargeted metabolomics.

Non-fermented teas, which are widely consumed in China, Japan, Korea, and elsewhere, have refreshing flavors and valuable health benefits. Various types of non-fermented teas look and taste similar and have no obvious differences in appearance, making their classification challenging. To date, there are very few reports about characterization and discrimination of different types of non-fermented teas. To characterize non-fermented teas and build a standard model for their classification based on their chemical composition, we employed multi-platform-based metabolomics to analyze primary and secondary metabolites in three main categories of non-fermented teas (green, yellow, and white), using 96 samples collected from China. Five hundred and ninety unique tea metabolites were identified and quantified in these three types of teas. Moreover, a partial least squares discriminant analysis (PLS-DA) model was established based on metabolomics data, in order to classify non-fermented teas into these three classes. Furthermore, our results speculate that the health benefits (e.g., antioxidant content) of these three types of non-fermented tea differ primarily because of variation in their metabolic components (e.g., ascorbate, vitexin).

Effects of nitrogen supply on flavonol glycoside biosynthesis and accumulation in tea leaves (Camellia sinensis).

Widely distributed in tea plants, the flavonoid flavonol and its glycosylated derivatives have important roles in determining tea quality. However, the biosynthesis and accumulation of these compounds has not been fully studied, especially in response to nitrogen (N) supply. In the present study, 'Longjing 43' potted tea seedlings were subjected to N deficiency (0g/pot), normal N (4g/pot) or excess N (16g/pot). Quantitative analyses using Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-QqQ-MS/MS) revealed that most flavonol glycosides (e.g., Quercetin-3-glucoside, Kaempferol-3-rgalactoside and Kaempferol-3-glucosyl-rhamnsoyl-glucoside) accumulated to the highest levels when treated with normal N. Results from metabolomics using Gas Chromatography-Mass Spectrometer (GC-MS) suggested that the levels of carbohydrate substrates of flavonol glycosides (e.g., sucrose, sucrose-6-phosphate, D-fructose 1,6-bisphosphate and glucose-1-phosphate) were positively correlated with flavonol glycoside content in response to N availability. Furthermore, Quantitative Real-time PCR analysis of 28 genes confirmed that genes related to flavonoid (e.g., flavonol synthase 1, flavonol 3-O-galactosyltransferase) and carbohydrate (e.g., sucrose phosphate synthase, sucrose synthase and glucokinase) metabolism have important roles in regulating the biosynthesis and accumulation of flavonol glycosides. Collectively, our results suggest that normal N levels promote the biosynthesis of flavonol glycosides through gene regulation and the accumulation of substrate carbohydrates, while abnormal N availability has inhibitory effects, especially excess N.

Metabolomic and transcriptional analyses reveal the mechanism of C, N allocation from source leaf to flower in tea plant (Camellia sinensis. L).

Tea flowering in late autumn competes for a large amount of nitrogen and carbohydrates, potentially undermines the storage of these resources in vegetative organs, and negatively influences the subsequent spring tea yield and quality. The mechanism underlying the re-allocation N and carbohydrate from source leaf to flower in tea plant has not been clearly understood. In this study, 15N allocation, changes in metabolomics, and gene expression in flower buds, flowers, and adjacent leaves were characterized. Total N content of the adjacent leaves significantly decreased during flowering while such a decrease could be reversed by flower bud removal. Foliar-applied 15N in the adjacent leaves markedly decreased and was readily allocated to flowers. Metabolomic analysis revealed that most sugars and benzoic acid increased by more than two-fold whereas theanine, Gln, Arg, Asp, and Asn decreased when flower buds fully opened to become flowers. In this process, Gly, Pro, and cellobiose in the adjacent leaves increased considerably whereas sucrose, galactose, benzoic acid, and many fatty acids decreased. Removal of flower buds reversed or alleviated the above decreases and led to an increase of Asn in the leaves. The expression of genes associated with autophagy (ATG5, ATG9, ATG12, ATG18), sucrose transporters (SUT1, SUT2, SUT4), amino acids permease (AAP6, AAP7, AAP8), glutamine synthetase (GS1;1, GS1;2, GS1;3), and asparagine synthetase (ASN1, ASN2) was significantly up-regulated in leaves during the flowering process and was strongly modulated by the removal of flower buds. The overall results demonstrated that leaves are the ready source providing N and carbohydrates in flowering and a series of genes related to autophagy, protein degradation, turn-over of amino acids, and phloem loading for transport are involved.

iTRAQ-Based Quantitative Proteomics Analysis Reveals the Mechanism Underlying the Weakening of Carbon Metabolism in Chlorotic Tea Leaves.

To uncover mechanism of highly weakened carbon metabolism in chlorotic tea (Camellia sinensis) plants, iTRAQ (isobaric tags for relative and absolute quantification)-based proteomic analyses were employed to study the differences in protein expression profiles in chlorophyll-deficient and normal green leaves in the tea plant cultivar "Huangjinya". A total of 2110 proteins were identified in "Huangjinya", and 173 proteins showed differential accumulations between the chlorotic and normal green leaves. Of these, 19 proteins were correlated with RNA expression levels, based on integrated analyses of the transcriptome and proteome. Moreover, the results of our analysis of differentially expressed proteins suggested that primary carbon metabolism (i.e., carbohydrate synthesis and transport) was inhibited in chlorotic tea leaves. The differentially expressed genes and proteins combined with photosynthetic phenotypic data indicated that 4-coumarate-CoA ligase (4CL) showed a major effect on repressing flavonoid metabolism, and abnormal developmental chloroplast inhibited the accumulation of chlorophyll and flavonoids because few carbon skeletons were provided as a result of a weakened primary carbon metabolism. Additionally, a positive feedback mechanism was verified at the protein level (Mg chelatase and chlorophyll b reductase) in the chlorophyll biosynthetic pathway, which might effectively promote the accumulation of chlorophyll b in response to the demand for this pigment in the cells of chlorotic tea leaves in weakened carbon metabolism.

Metabolic Changes of Amino Acids and Flavonoids in Tea Plants in Response to Inorganic Phosphate Limitation.

The qualities of tea (Camellia sinensis) are not clearly understood in terms of integrated leading molecular regulatory network mechanisms behind inorganic phosphate (Pi) limitation. Thus, the present work aims to elucidate transcription factor-dependent responses of quality-related metabolites and the expression of genes to phosphate (P) starvation. The tea plant organs were subjected to metabolomics analysis by GC×GC-TOF/MS and UPLC-Q-TOF/MS along with transcription factors and 13 metabolic genes by qRT-PCR. We found P starvation upregulated SPX2 and the change response of Pi is highly dependent on young shoots. This led to increased change in abundance of carbohydrates (fructose and glucose), amino acids in leaves (threonine and methionine), and root (phenylalanine, alanine, tryptophan, and tyrosine). Flavonoids and their glycosides accumulated in leaves and root exposed to P limitation was consistent with the upregulated expression of anthocyanidin reductase (EC 1.3.1.77), leucoanthocyanidin dioxygenase (EC 1.4.11.19) and glycosyltransferases (UGT78D1, UGT78D2 and UGT57L12). Despite the similar kinetics and high correlation response of Pi and SPX2 in young shoots, predominating theanine and other amino acids (serine, threonine, glutamate, valine, methionine, phenylalanine) and catechin (EGC, EGCG and CG) content displayed opposite changes in response to Pi limitation between Fengqing and Longjing-43 tea cultivars.

Lipidomics analysis unravels the effect of nitrogen fertilization on lipid metabolism in tea plant (Camellia sinensis L.).

BACKGROUND: Nitrogen (N) plays an important role in the formation of tea quality-related compounds, like amino acids and flavor/aroma origin compounds. Lipids, which have been reported to be affected by N deficiency, are precursors to the generation of flavor/aroma origin compounds in tea plant. However, there is no literature about the lipid profiles of tea plant affected by N fertilization. Hence, we hypothesize that the biosynthesis of flavor-related compounds in tea was affected by N through its regulation of lipid metabolism. RESULTS: In this study, mature leaves and new shoots of tea plant grown under three N levels at the rates of 0, 285 and 474 kg/ha were applied for ultra-performance liquid chromatography-mass spectrometry (UPLC/MS) based lipidomic analysis. Totally, 178 lipid species were identified. The results showed that the composition of lipid compounds in mature leaves and new shoots varied dramatically, which was also affected by N levels. The higher content of the storage lipid TAG and higher carbon (C)/N ratio in mature leaves than that of new shoots in tea plants grown under low N level (0 kg/ha) suggested that tea plants could remobilize the C stored in TAG to maintain their C/N balance and help to improve the quality of tea. N fertilization resulted in a higher content of the compounds 36:6 MGDG and 36:6 DGDG. Since these compounds contain linolenic acid (18:3), a precursor to the formation of aroma origin compounds, we suggested their increase could contribute to the quality of tea. CONCLUSIONS: Taken together, the present work indicated that appropriate application of N fertilizer could balance the lipid metabolism and the formation of flavor/aroma origin compounds, which help to improve the quality of tea. Moreover, excess N fertilization might deteriorate the aroma quality of made tea due to increases of precursors leading to grassy odor.

Metabolomics analysis reveals the metabolic and functional roles of flavonoids in light-sensitive tea leaves.

BACKGROUND: As the predominant secondary metabolic pathway in tea plants, flavonoid biosynthesis increases with increasing temperature and illumination. However, the concentration of most flavonoids decreases greatly in light-sensitive tea leaves when they are exposed to light, which further improves tea quality. To reveal the metabolism and potential functions of flavonoids in tea leaves, a natural light-sensitive tea mutant (Huangjinya) cultivated under different light conditions was subjected to metabolomics analysis. RESULTS: The results showed that chlorotic tea leaves accumulated large amounts of flavonoids with ortho-dihydroxylated B-rings (e.g., catechin gallate, quercetin and its glycosides etc.), whereas total flavonoids (e.g., myricetrin glycoside, epigallocatechin gallate etc.) were considerably reduced, suggesting that the flavonoid components generated from different metabolic branches played different roles in tea leaves. Furthermore, the intracellular localization of flavonoids and the expression pattern of genes involved in secondary metabolic pathways indicate a potential photoprotective function of dihydroxylated flavonoids in light-sensitive tea leaves. CONCLUSIONS: Our results suggest that reactive oxygen species (ROS) scavenging and the antioxidation effects of flavonoids help chlorotic tea plants survive under high light stress, providing new evidence to clarify the functional roles of flavonoids, which accumulate to high levels in tea plants. Moreover, flavonoids with ortho-dihydroxylated B-rings played a greater role in photo-protection to improve the acclimatization of tea plants.